B cell antibody responses are triggered by the binding of antigen to the clonally distributed B cell antigen receptors (BCRs). Over the last several years a great deal has been learned about the biochemistry of the complex signal cascades triggered by BCR antigen engagement. However, what remains relatively poorly understood are the molecular events that trigger the initiation of signaling. Using live cell TIRF microscopy, we showed that in B cells responding to antigen presented on a planar lipid bilayer, simulating an antigen presenting cell, BCRs form signaling active microclusters by a mechanism that does not require physical crosslinking of BCRs by multivalent antigens. We observed that in response to either monovalent or multivalent antigens the BCRs accumulate and form microclusters at the initial points of contact of the B cell membrane with the bilayer. Clustering did not depend on the ability of the BCR to signal subsequently, revealing clustering to be an intrinsic property of the BCRs. The microclusters grew by trapping mobile BCRs and the larger clusters were actively organized into an immune synapse. The kinetics of these events and signaling were identical for monovalent and multivalent antigens. We determined that the membrane-proximal ecto-domain of the BCR mIg was both necessary and sufficient for BCR oligomerization and signaling. BCRs that contained a mIg in which Cmu4 was deleted failed to cluster and to signal when engaging monovalent antigen. Conversely, Cmu4 expressed alone on the B cell surface spontaneously clustered and activated B cells. These findings lead us to propose a novel mechanism by which BCRs form signaling active microclusters that we termed the conformation induced oligomerization model for the initiation of BCR signaling. According to our model BCR in the resting state are not in an oligomerization receptive conformation such that random bumping has no repercussion. The binding of antigen on an opposing membrane exerts a force on the BCR to bring it into an oligomerization receptive form so that when two antigen-bound BCRs bump they oligomerize. An important prediction from our model is that BCRs exist mostly as monomers on B cell surfaces and that Ag induces their clustering to initiate signaling. However, evidence for an opposing model has emerged, namely that BCRs are organized into clusters on resting cell surfaces and that Ag serve to disassociate these clusters to initiate signaling. Through a collaboration with Dr. Lippincott-Schwartz at the NIH, a leader in the development of methods to quantify the spatial organization of surface receptors by point-localization super resolution imaging using pair correlation analysis, we have now obtained robust evidence that the vast majority of IgM BCRs expressed by human peripheral blood naive B cells exist as monomers in the resting state. To determine if the organization of the BCRs on the B cell surface varied with the differentiated state of the B cell, we analyzed the distribution of IgG BCRs on human peripheral blood memory B cells. Our previous studies provided evidence for intrinsic differences in IgM- and IgG-BCRs that contributed to the more efficient initiation of B cell activation by IgG BCRs. These findings lead us to hypothesize that IgG BCRs may also be spatially organized on resting cells to facilitate rapid clustering upon antigen binding. However, we determined that IgG BCRs were monomers on the surface of resting memory B cells and that their organization did not differ significantly from that of IgM BCRs on naive cells. We observed that upon engagement of Ag both IgM and IgG BCRs formed large clusters containing hundreds of BCRs although the IgG BCR clusters on memory B cells formed more rapidly and reached larger sizes as compared to IgM BCRs on naive B cells, consistent with our previous data showing that antigen-driven BCR clustering is more efficient for IgG-BCR-versus IgM-BCR-expressing cells. Over the last year we also begun a new initiative to characterize BCR signaling and antigen-internalization in human tonsillar germinal center (GC) B cells. GCs are compartments within secondary lymphoid organs in which in response to Ag, B cell clonal expansion, somatic hypermutation and affinity-based selection occur resulting in the production of isotype-switched memory B cells and high affinity antibody secreting plasma cells. In recent years discrete steps in GC reactions have been mapped out in mouse models. However, our understanding of the B cell biology of human GCs lags behind the mouse models and clearly knowledge of the cellular and molecular mechanism by which high affinity memory B cells and plasma cells are generated would aid in vaccine design. In collaboration with Dr. Susan Moir (LIR, NIAID), we devised a cell separation strategy that provided highly pure populations of tonsil naive B cells, memory B cells and GC dark and light zone B cells. Our analysis thus far indicates that the GC B cells express less BCR per cell as compared to either naive or memory B cells and are less active in clustering their BCR and reorganizing the actin cytoskeleton to form immune synapses. GC B cells also showed a remarkably higher affinity threshold for the internalization of antigen that we correlated with alterations in the actin cytoskeleton. We plan to correlate the outcome of BCR antigen engagement in naive, GC and memory B cells with transcriptional activation and alterations in cellular metabolism. It is our hypothesis that the outcome of BCR signaling in these B cell populations will differ and that these differences will shed light on the function of these B cell subpopulations.